In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of an illuminating light beam is moved in a sample plane using a controllable beam deflection device, generally by tilting two mirrors; the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by galvanometer positioning elements. The power of the light coming from the sample is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to determine the current mirror position.
In confocal scanning microscopy specifically, a sample is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally includes a light source, a focusing optical system used to focus the light of the source onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detection or fluorescent light. The illuminating light is coupled in, for example, via a beam splitter. The fluorescent or reflected light coming from the sample travels back via the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that point information is obtained which leads to a three-dimensional image by sequential scanning of the sample.
In order to couple the excitation light of at least one light source into the microscope and to separate out, from the light coming via the detection beam path from the sample, the excitation light scattered and reflected at the sample, or the excitation wavelength, it is also possible to provide, instead of the beam splitter, an optical device embodied as an acousto-optical element, for example as known from German Unexamined Application DE 199 06 757 A1.
A three-dimensional image is usually achieved by acquiring image data in layers; the path of the scanning light beam on or in the sample ideally describing a meander (scanning one line in the x-direction at a constant y-position, then stopping the x-scan and slewing by y-displacement to the next line to be scanned, then scanning that line in the negative x-direction at a constant y-position, etc.). To allow the acquisition of image data in layers, the sample stage or the objective lens is shifted after a layer has been scanned, and the next layer to be scanned is thus brought into the focal plane of the objective lens.
In some microscopic applications, it is necessary to be able to manipulate the sample during scanning or between two scanning operations. Such manipulation may include, for example, the release of bound dyes, a bleaching operation, a cutting operation, or the use of optical tweezers.
U.S. Pat. No. 6,094,300 describes a laser scanning microscope including a first light source whose light is scanned over a sample by a first scanner, and further including a second light source whose light can be scanned over the sample as manipulation light by a second scanner.
German Patent Application DE 100 39 520 A1 also discloses a scanning microscope including two beam deflection devices, which independently scan light from different light sources over or through a sample.